Yuki Sato, Ramjay Vatsan, Bharat H. Joshi, Syed R. Husain* and Raj K. Puri Pages 758 - 770 ( 13 )
<p>Background: Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cellmediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors <i>in vivo</i>, indicating that host immune responses against IL-13Rα2 need further augmentation. <p> Objective: The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing <i>IL-13Rα2</i> (rMVA-IL13Rα2) virus and study <i>in vitro</i> infectivity and efficacy against IL-13Rα2 positive cell lines. <p> Methods: We constructed a recombinant MVA expressing <i>IL-13Rα2</i> and a green fluorescent protein (<i>GFP</i>) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. <p> Results: Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP<sup>+</sup> fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13- PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. <p> Conclusion: rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.</p>
MVA, modified vaccinia ankara, IL-13Rα2, interleukin-13 receptor α2, GFP, green fluorescent protein, IL13-PE, interleukin-13 fused to mutated form of <i>Pseudomonas exotoxin</i>.